Fucosylated mammalian glycans have functions in fertilization (1), early differentiation of embryo (2), brain development (3,4), and leukocyte extravasation (5,6). the α1-3/4fucosylated oligo- and polysaccharides being conjugated to lipids and proteins or as free oligosaccharides such as the oligosaccharides of human milk. The fucosylated N-acetyllactosamines (Lewis x, Galβ1-4(Fucα1-3)GlcNAc and Lewis a, Galβ1-3(Fucα1-4)GlcNAc) and fucosylated N-acetyllactosdiamine (LexNAc, GalNAcβ1-4(Fucα1-3)GlcNAc) occur often as terminal sequences such as Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα-, Galβ1-3)(Fucα1-4)GlcNAcβ1-2Manα-, GalNAcβ1-4(Fucα1-3(GlcNAcβ1-2Manα-, Galβ1-4(Fucα1-3)GlcNAcβ1-3(Galβ-/GalNAcα-, Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ- , Galβ1-4(Fucα1-3) GlcNAcβ1-4Manα-, Galβ1-3(Fucα1-4(GlcNAcβ1-4Manα-, and Galβ1-4(Fucα1-3)GlcNAcβ1-6Galβ-/GalNAcα-/Manα-. In the middle of lactosamine-type chains the Lewis x-sequences, -GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Gal are common, but also Lewis a-structures, Galβ1-3(Fucα1-4)GlcNAc, are present with a possibility for repeating the sequence chain. In the free oligosaccharides found mainly in human milk, the α1-3fucosylated epitope at the reducing end of the saccharide is commonly lactose (Galβ1-4Glc) or its elongated/substituted form such as Galβ1-4(Fucα1-3)Glc, Fucα1-2Galβ1-4(Fucα1-3)Glc, Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc, Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3(Glc, and Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc. Analogs of these could be useful for studies of specificities of biological activities of the natural mammalian α1-3/4fucosylated sequences.
The fucosylation step in the biosynthesis of these glycans is accomplished by the family of α1-3/4fucosyltransferases (Fuc-Ts). In man, at least Fuc-Ts III-VII and IX are expressed (7-9), and enzymatically active homologs are known in other animals and bacteria.
α1-3Fucosyltransferases transfer fucose to position 3 of GlcNAc or Glc residues in Galβ1-4GlcNAc (LacNAc), GalNAcβ1-4GlcNAc (LacdiNAc) and Galβ1-4Glc (lactose) to synthesize the bioactive epitopes Galβ1-4(Fucα1-3)GlcNAc (Lewis x, Lex), GalNAcβ1-4(Fucα1-3)GlcNAc (LexNAc), and Galβ1-4(Fucα1-3)Glc, respectively. All human Fuc-Ts are known to use Galβ1-4GlcNAc-type acceptors (7,9). GalNAcβ1-4GlcNAc serves also as an acceptor for the Fuc-Ts of human milk (10). Human Fuc-Ts III and V have also α1-4fucosyltransferase activity using acceptors such as Galβ1-3GlcNAc (type I N-acetyl-lactosamine) to synthesize Galβ1-3(Fucα1-4)GlcNAc (Lewis a). At least human Fuc-Ts III and V and (weakly VI) are able to fucosylate lactose and related oligosaccharides to structures containing Galβ1-4(Fucα1-3)Glc sequences.
Enzymatic α1-3fucosylation of N-acetyl-chitobiose has been described, se U.S. Pat. No. 5,759,823, but the product was not characterized. The reducing N-acetyl-chitobiose contains also the epimer in which there is ManNAc at the reducing end, and it is not known from the data of the said U.S. patent if N-acetyl-chitobiose or its reducing-end epimer was fucosylated. Saccharides with fucosylated reducing-end GlcNAc are not considered useful as they are labile and degrade even in aqueous solutions at near neutral pH. The reducing-end fucosylated GlcNAc is very rate, or non-existent, in mammalian natural oligosaccharides, possibly because of the lability which could make it useless also in vivo for biological functions.
The present invention describes saccharide epitope analogues of the mammalian fucosylated saccharide chains, as well as their synthesis. An effective method to synthesize such epitopes is to use α1-3 fucosyltransferases or α1-3/4fucosyltransferases for fucosylation of novel acceptor sequences. Some of the acceptor sequences can be synthesized from cheap natural polysaccharides such as cellulose, chitin, chondroitin/chondroitin sulphates, or hyaluronic acid, also natural polysaccharides with the sequence Glcβ1-(3Glcβ1-4Glcβ1-)□3Glc could be used to synthesize acceptors. β1-4GlcNAc transferase and UDP-GlcNAc can be used to generate GlcNAcβ1-4GlcNAcβ1- linked to Gal, GlcNAc or Man (11) and these can be used to make other analogues. Certain parasites have also been reported to contain N-acetyl-chitooligosaccharides which could be used as acceptors for the fucosylation reaction (12).
Interestingly the novel fucosylations of N-acetyl-chitooligosaccharides (N-acetyl-chitotriose and larger) described here occured to the non-reducing subterminal residue (forming a terminal Lewis x-like structure with a linkage structure similar to human glycans) and not to the reducing-end GlcNAc as in plant N-glycans.